The present invention is generally in the area of drug delivery devices and is specifically in the area of polymeric drug delivery devices.
Gene therapy is generally defined as the introduction and expression of an exogenous gene in an animal to supplement or replace a defective or missing gene. Examples that have received a great deal of recent attention include the genes missing in cystic fibrosis and severe combined immunodeficiency. Although tremendous progress has been made in the area of gene therapy, obtaining long term expression of the desired proteins remains elusive.
In the majority of cases, a retroviral vector is used to introduce the gene to be expressed into appropriate cells. Gene transfer is most commonly achieved through a cell-mediated ex vivo therapy in which cells from the blood or tissue are genetically modified in the laboratory and subsequently returned to the patient. The clinical studies by Steven Rosenberg, et al., "Immunotherapy of patients with metastatic melanoma using tumor-infiltrating lymphocytes and IL-2", Preliminary report, New England J. Med., 319 (1988) 1676-1680, using in vitro-activated LAK and TIL for tumor destruction illustrates this approach. In other cases, the vector carrying the gene to be expressed is introduced into the patient, for example, by inhalation into the lungs in the case of cystic fibrosis. Transfected cells have also been implanted, alone or encapsulated within a protective membrane that protects the cells from the inflammatory response of the body while at the same time allowing the gene product to diffuse out of the membrane. There have also been reports of the direct injection of an exogenous gene in combination with an appropriate promoter, into tissue, with some transient expression being noted.
Viral vectors have been widely used in gene transfer, due to the relatively high efficiency of transfection and potential long term effect through the actual integration into the host's genome. However, there are still concerns about the risks involved in the use of viruses. Activation of proto-oncogenes and reversion to wild-type viruses from replication incompetent viruses are some important potential hazards of viral delivery of genes.
Since the discovery that naked DNA is taken up by muscle cells and transiently expressed in vivo, and subsequent reports, by Wolff, Jon A et al., "Direct gene transfer into mouse muscle in vivo," Science, 247, 1465-1468, 1990; and Acsadi et al., "Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs," Nature, 352, 815-818, 1991, there has been increasing interest in using non-viral vehicles for in vivo transfections.
Plasmid DNA, which can function episomally, has been used with liposome encapsulation, CaPO.sub.4 precipitation and electroporation as an alternative to viral transfections. Recent clinical trials with liposome encapsulated DNA in treating melanoma illustrates this approach to gene therapy, as reported by Nabel, J. G., et al., "Direct gene transfer with DNA-liposome complexes in melanoma: Expression, biological activity and lack of toxicity in humans", Proc. Nat. Acad. Sci. U.S.A., 90 (1993) 11307-11311. A foreign gene coding for HLA-B was introduced into subcutaneous sites of melanoma tumors. Expression of the new gene and the absence of an anti-DNA host response was confirmed. Wolff, Jon A, "Persistence of plasmid DNA and expression in rat brain cells in vivo," Experimental Neurology, 115, 400-413, 1992, also reported expression of plasmid DNA. Thus, direct gene transfer offers the potential to introduce DNA encoding proteins to treat human diseases.
The mechanisms for cellular uptake of exogenous DNA and subsequent expression are not clear but gene transfer with naked DNA is associated with several characteristics. Unlike in the case of oligonucleotides, which are typically a maximum of twenty to thirty nucleotides in length, genes encoding most molecules of therapeutic interest are quite large, and therefore considerably more difficult to introduce into cells other than through retroviral vector, or in vitro, by chemical manipulation, so that the efficiency of transfer is low. In most reported cases to date, only transient expression of up to a few weeks or months has been observed. Although low level expression and short term expression are two important drawbacks with direct DNA transfer, transfections with naked DNA have several advantages over viral transfers. Most importantly, concerns related to the immunogenicity and transforming capability of viruses are avoided. In addition, naked DNA is easy to produce in large quantities, is inexpensive, and can be injected at high concentration into localized tissue sites allowing gene expression in situ without extensive ex vivo therapy.
The following additional articles review the current state of gene therapy and the problems associated therewith: Blau, Helen M, "Muscling in on gene therapy," Nature, 364, 673-675, 1993; Cohen, Jon, "Naked DNA points way to vaccines," Science, 259, 1691-1692, 1993; Dagani, Ron, "Gene therapy advance, anti-HIV antibodies work inside cells," C&EN, 3-4, 1993; Felgner, Philip L, "Lipofectamine reagent: A new, higher efficiency polycationic liposome transfection reagent," Focus/Gibco, 15, 73-78, 1993; Liu, Margaret A et al., "Heterologous protection against influenza by injection of DNA encoding a viral protein," Science, 259, 1745-1749, 1993; Marx, Jean, "A first step toward gene therapy for hemophilia B," Science, 262, 29-30, 1993; Mulligan, Richard C, "The basic science of gene therapy," Science, 260, 926-931, 1993; Nicolau, Claude et al., "In vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin I," Proc. Natl. Acad. Sci. USA, 80, 1068-1072, 1983; Partridge, Terence A, "Muscle transfection made easy," Nature, 352, 757-758, 1991; Wilson, James M, "Vehicles for gene therapy," Nature, 365, 691-692, 1993; Wivel, et al., "Germ-line gene modification and disease prevention: Some medical and ethical perspectives," Science, 262, 533-538, 1993; and Woo, Savio L C et al., "In vivo gene therapy of hemophilia B: sustained partial correction in Factor IX-deficient dogs," Science, 262, 117-119, 1993.
Gene therapy is one of the most promising areas of research today. It would therefore be extremely useful if one had an efficient way to introduce genes into cells which yielded long term expression.
It is therefore an object of the present invention to provide a means for efficient transfer of exogenous genes to cells in a patient.
It is a further object of the present invention to provide a means for long term expression of exogenous genes in patients.
It is a further object of the present invention to provide a means for increasing or decreasing the inflammatory response to implanted polymeric devices.
It is a still further object of the present invention to provide a method for immunization of individuals over a more prolonged period of time than is achieved by a single or multiple immunization protocol.
It is another object of the present invention to provide a method for targeting of gene delivery either to tissue cells or to inflammatory type cells.